Brandon:Protocols/Cloning Transformation3: Difference between revisions

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(Created page with "==cloning and A-tailing etc.== 1. reaction from the manual transfect into E. coli, use transfection protocol 1. Thaw one vial of One Shot TOP10 chemically competent cel...")
 
>Bsos
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==cloning and A-tailing etc.==
==cloning and A-tailing etc.==


1. reaction from the manual
*procedure for TA cloning and tranformation of E. Coli




transfect into E. coli, use transfection protocol
===Cloning into vector===


*make sure you know to use TA cloning or blunt cloning vector.
*procedure for cloning with Invitrogen TA cloning kit, for TA cloning.
1. amount of PCR product needed. = ((200)(50))/3900 = 2.5 ng
2. reaction mixture
    3 uL PCR product
    1 uL ligation buffer
    2 uL pCR vector 2.1 (25 ng/ul)
    1 uL nuclease free water
    3 uL T4 DNA ligase  (used 1U/uL T4 not from kit, Kit T4 is @ 5U/uL
    10 uL total
3. incubate at 14C for 4 hours or overnight preferably
4. Now ready for transformation into E coli.
===Transformation of E. coli===
*transfect into E. coli, use transfection protocol


  1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
  1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
     Add 1-3 uL of PCR product to TOP10 cell vial (uL based on calculation)
     Add 1-3 uL of PCR product/ligated cloning vector from above to TOP10 cell vial (uL based on calculation),
     MIX GENTLY, no pipetting up and down
     MIX GENTLY, no pipetting up and down
   
   

Revision as of 00:08, 16 May 2012

cloning and A-tailing etc.

  • procedure for TA cloning and tranformation of E. Coli


Cloning into vector

  • make sure you know to use TA cloning or blunt cloning vector.
  • procedure for cloning with Invitrogen TA cloning kit, for TA cloning.
1. amount of PCR product needed. = ((200)(50))/3900 = 2.5 ng
2. reaction mixture
    3 uL PCR product
    1 uL ligation buffer
    2 uL pCR vector 2.1 (25 ng/ul)
    1 uL nuclease free water
    3 uL T4 DNA ligase  (used 1U/uL T4 not from kit, Kit T4 is @ 5U/uL
   10 uL total

3. incubate at 14C for 4 hours or overnight preferably

4. Now ready for transformation into E coli.


Transformation of E. coli

  • transfect into E. coli, use transfection protocol
1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
    Add 1-3 uL of PCR product/ligated cloning vector from above to TOP10 cell vial (uL based on calculation),
    MIX GENTLY, no pipetting up and down

2.  incubate vials on ice for 30 minutes

3.  Heat-shock cells for 30 seconds at 42C, no shaking

4.  remove from 42C and place on ice for 2 minutes

5.  add 250 ul pre-warmed SOC medium to each vial

6.  cap vials tightly and shake horizontally at 37C for 1 hr at 224 rpm in shaking incubator

7.  spread 20-200 ul of transformations onto pre-warmed selective plates and incubate overnight at 37C.
    *use 2-3 different concentrations, 1:10, 50 uL, 200 uL or w/e

8.  Store reamining transforamtion mix at 4C. cells can be plated next day.

9.  Invert selective plates, incubate at 37 overnight.

10. Select colonies and analyze with PCR or w/e.